Mammalian hepatic microsomes contain a membrane bound enzyme system which catalyzes diverse mixed-function oxidations. This microsomal mixed-function oxidase enzyme system metabolizes a variety of hydrophobic foreign compounds and hormones, and requires substrate, NADPH, and molecular oxygen for activity. The mixed-function oxidase hemoprotein cytochrome P-450 or P-488 may bind hydrophobic substrates and effectors of mixed-function molecules. Although many nitrogen containing substrates and effectors of mixed-function oxidase activity bind to these cytochromes, no systematic study has characterized the reactive site(s) involved in the binding of nitrogenous ligands and their effects on mixed-function oxidase activity. This would be important since man is exposed to many of these nitrogenous compounds in the form of air pollutants, food additives and drugs. I propose to use a reconstituted mixed-function oxidase system containing purified cytochrome P-450 or P-448 to study the ability of nitrite, organonitrates, and nitrosamines to: a) bind cytochrome P-450 or P-448 and alter their absorption spectra: B9 compete with known ligands (carbon monoxide, oxygen, metyrapone) for binding; c) affect the binding of hydroxylation substrates (naphthalene, aniline) and demethylation substrates (aminopyrine, p-nitroanisole); d) affect the mixed-function oxidase activity using these substrates; and e) influence the effects of sulfhydryl reagents, sulfhydryl inhibitors, and free radical scavangers on enzyme activity and substrate binding. The results obtained from this study, when taken together, should reveal the location the characteristics, and the consequences of binding various nitrogenous ligands to cytochrome P-450 or P-488. This multifaceted approach has not been attempted previously.